This research project will investigate the ultrastructural details of receptor mediated endocytosis of the major cell surface antigens present on human T (CD2, CD3, CD4, CD5, CD8, CD25), B (CD19, CD20, surface immunoglobulin, DR) and NK (CD11, CD16) lymphocytes. To achieve this objective, immunoperoxidase and immunogold electron microscopy will be employed to follow the pathways of internalization of murine monoclonal antibodies and immunotoxins bound to these surface receptors. Specific histochemical reactions will be utilized to distinguish clathrin-associated pits and vesicles, acidic endosomal compartments, Golgi, and lysosomes. The findings of these morphologic studies will be independently verified by experiments studying the progression of radiolabeled monoclonal antibodies and immunotoxins from the cell surface through the various intracellular compartments by the techniques of subcellular fractionation of disrupted cells and ultracentrifugation on Percoll gradients. The position of various organelles in such fractions will be ascertained by buoyant density determination, electron microscopy, and marker enzyme analysis. The patterns of cell surface antigen endocytosis revealed in the above studies will be correlated with the efficacy of immunotoxins targeted to the different receptors in four model systems in an attempt to discern the patterns of internalization associated with greatest cell killing. These experiments are designed to elucidate the ultrastructural characteristics which underlie the variable efficacy which has been observed with synthesized immunotoxins. A complete comprehension of the determinants of immunotoxin efficacy could represent a major advance in the design of potent, specific anti-cancer reagents. Furthermore, the studies outlined in this proposal should be of general interest for the field of basic immunology since the lymphocyte surface antigens studied mediate important recognition and cell activation functions (e.g. the "T3"-T cell receptor complex, the E rosette receptor, surface immunoglobulin, the T gamma receptor), and delineation of the behavior of these molecules following ligand binding will advance our understanding of the fundamental immunologic processes mediated by these molecules.